CN103060455B - A kind of helicobacter pylori infections individualized treatment detection gene chip and application - Google Patents

Abstract

The present invention relates to a kind of biological engineering class detection product and application thereof.The specifically detection type gene chip of a kind of helicobacter pylori infections individualized treatment, this chip can detect helicobacter pylori clarithromycin resistant mutational site A2142G with A2143G, mediated quinolone resistance mutational site Asn87 (N87K) 2C19*2 (G6981A) relevant with proton pump inhibitor metabolism in human cytochrome enzyme CYP450 with Asp91 (D91G/Y/N) and the polymorphism in 2C19*3 (G636A) site simultaneously.This chip detection method is quickly, accurately, the qualification of helicobacter pylori cause of disease, clarithromycin and mediated quinolone resistance analysis and the analysis of proton pump inhibitor metabolism individuation difference can be carried out, be used for the personalized medicine instructing helicobacter pylori infections " three furnace process " to treat.

Description

A kind of helicobacter pylori infections individualized treatment detection gene chip and application

Technical field

The present invention relates to a kind of helicobacter pylori infections individualized treatment detection gene chip and application, the technology related to is led Territory is medical test and biochip technology.It it is a kind of detection type gene core for helicobacter pylori infections individualized treatment Sheet, can be simultaneously to human genome CYP4502C19*2, CYP4502C19*3 polymorphism and helicobacter pylori clarithromycin, quinolinones Class drug resistance site is detected.

Background technology

Helicobacter pylori (H.pylori, HP) is the I class cancerigenic factor that WHO international cancer research institution determines.China HP Infection rate is 40%-90%, and average out to 59% belongs to the severely afflicated area of HP relevant disease.HP infect be cause chronic gastritis and Cause peptic ulcer occur and recurrence major reason, and with low potential malignancy gastric mucosa-associated lymphoid tissue (MALT) lymphoma Generation, develop closely related.Research shows, helicobacter pylori infections is the cause of disease that gastric cancer occurs, the root to helicobacter pylori Except the occurrence risk that can obviously reduce crowd's gastric cancer.But HP drug resistance is a global problem, it is the master of HP radical rate decline Want reason.Combine amoxicillin and the triple therapy of clarithromycin (or metronidazole) with proton pump inhibitor (PPI), be domestic and international The a line anti-HP treatment of infection combination generally acknowledged^[6].Quinolones is the Second line Drug of HP radical cure.The curative effect master of anti-HP treatment To be affected by following two factor: one is the abuse due to antibiotic in recent years, clarithromycin, metronidazole, mediated quinolone resistance Constantly rise so that clarithromycin or the effective percentage of quinolones in triple therapy decline；Another one affects three The important factor in order of therapy medication success rate is the use of PPI inhibitor.PPI metabolism and human-cytochrome P450 (CYP450) Isozyme CYP2C19 is closely related, and the gene pleiomorphism type of CYP2C19 determines PPI accretion rate (strong generation in human body Thank, middle metabolism and weak metabolism)^[7；8].Therefore, before using triple therapy, if it is possible to clarithromycin or first accurately are provided Nitre azoles, quinolinones drug sensitivity tests and the type of CYP2C19, select medicine to carry out personalized medicine pointedly, then ensure that Treatment success rate, reduces untoward reaction and the wasting of resources that abuse of antibiotics brings simultaneously.

HP clarithromycin, mediated quinolone resistance and people's CYP450 gene pleiomorphism are mainly due to the mononucleotide of related gene Polymorphism (SNP) causes.For the detection of gene type, traditional cultivation and susceptibility method, to laboratory and operator Requirement high, experiment time-consuming long (about 1 week), greatly, and said method can not provide people CYP450 polymorphism information to workload, because of This limits its application in practice.The method for quick such as PCR are highly sensitive, high specificity, but detection flux is relatively low, and one Secondary being only capable of detects 2-3 target spot, can not fully meet clinical practice needs.Equally, although sequencing technologies is for we providing Detection method easily and efficiently, but it needs expensive instrument, and also detection sensitivity is the most far away not as good as chip technology.Gene Chip technology is a kind of sequencing, the genomic organization of scale and the new technique of expression study occurred in recent years, can be same The many sites of Shi Jinhang single-gene or the many sites of polygenes polymorphism typing detection, have contain much information, highly sensitive, parallel soon The features such as speed detection.Once previous research work completes to obtain gene chip product (detection probe and the hybridization bar of maturation Part determines), the sequencing that once thousands of allele are carried out gene type can be realized and routinize, this is other The superiority place that genotyping technique is incomparable.

Gene chip (Gene Chip) is that substantial amounts of oligonucleotide molecules is fixed on solid phase carrier formation DNA microdot Battle array, the sequence information of DNA sample is efficiently understood and analyzes by the characteristic by making nucleic acid molecular hybridization pairing.DNA chip Preparation method mainly has two kinds: one is that directly on stationary plane, in-situ chemical synthesizes a series of oligonucleotide, another kind be by Pre-synthesis different oligonucleotide according to certain arrangement mode point sample, be fixed on sulidus face.The chip prepared leads to Hybridization can be carried out after crossing chemical treatment.Pathogenic microorganism, progressively measures including antibacterial, viral gene order research, Providing a good point of penetration for its analysis, two gene chip has provided for detection and parallel study pathogenic microorganism again The instrument of power.The diagnosis prediction that biochip technology is used for disease has become the focus of whole world research, and has succeeded in developing inspection Survey the gene chip of the microorganisms such as part antibacterial.Biochip technology has following side for the advantage of disease diagnosing and predicting Face: one is the sensitive and accuracy of height, few with sample；Two be fast and convenient, the time is short；Three can detect many people simultaneously Multiple disease.In view of biochip technology has huge theory significance and actual application value, some research strength of China are relatively Rich unit has carried out the research work of this respect.But, how at the early-stage domestic practical chip research is, so far, There is not yet the rapid gene detection chip for the helicobacter pylori infections individualized treatment for population of China both at home and abroad.

Summary of the invention

The present invention is to set up a kind of detection type gene chip for helicobacter pylori infections individualized treatment, this chip Human genome CYP4502C19*2, CYP4502C19*3 polymorphism and pylorus spiral shell can be detected in once experiment without cultivating simultaneously Bacillus clarithromycin, quinolones drug resistance site.By Analysis and Screening human genome CYP4502C19*2, CYP4502C19*3's DNA and helicobacter pylori clarithromycin and quinolones drug resistance site DNA sequence, separately design different oligonucleotide fragments, Establish include detection and identify the comprehensive analyzing detecting method including biochip technology, to reaching quick, accurate, special Testing goal, and apply.

The whole world has more than the people of half and infects HP, and 80%-90% the infected has clinical symptoms, 10%-15% to develop into disappear Peptic-ulcer, 1%-2% develops into malignant tumor.Helicobacter pylori has been widely accepted with the relation of digestive tract disease, right HP carries out eradicating and can cure its disease caused, and is avoided that and develops into more serious disease, has great social application Prospect.

Present invention is generally directed to helicobacter pylori infections individualized treatment, by Analysis and Screening human genome The DNA of CYP4502C19*2, CYP4502C19*3 and helicobacter pylori clarithromycin and quinolones drug resistance site DNA sequence, Separately design different oligonucleotide fragments, establish include detection and identify and comprehensively analyze detection including biochip technology Method, to reaching quick, accurate, special testing goal, and is applied.Human genome can be provided except developing simultaneously CYP4502C19*2, CYP4502C19*3 polymorphism and helicobacter pylori clarithromycin, the gene chip of mediated quinolone resistance information, Also establish theory and technology basis for the molecular Biological Detection of more bacterial disease and qualification and anti-system.

A kind of helicobacter pylori infections individualized treatment detection gene chip, it is characterised in that chip includes (1) pylorus spiral shell Wild type corresponding for clarithromycin resistant mutational site A2142G with A2143G on bacillus 23s gene and saltant type SNP typing Oligonucleotide probe；(2) mediated quinolone resistance mutational site Asn87 (N87K) on helicobacter pylori gyrA gene and Asp91 (D91G/Y/N) corresponding wild type and saltant type SNP parting oligonucleotide probe；(3) proton in human cytochrome enzyme CYP450 Wild type that 2C19*2 (G6981A) that pump inhibitor metabolism is relevant and 2C19*3 (G636A) is corresponding and saltant type SNP typing widow Nucleotide probe；(4) chip quality controls oligonucleotide probe；(5) oligonucleotide probe solidification is formed on a support material Probe array.

So-called helicobacter pylori infections individualized treatment detection gene chip refers to detect people's CYP450 metabolism phase simultaneously The gene type of correlation gene 2C192,2C193 and helicobacter pylori are to clarithromycin and the Resistant genetype of quinolones Oligonucleotide arrays；

So-called oligonucleotide probe refers to and the poly oligonucleotide of genes of interest hybridization, ten to five ten alkali of length Base；

So-called quality control probes at least includes two kinds of probes, i.e. positive controls, negative control: positive control is used for examining Surveying whether normal hybridisation and being accurately positioned, negative control is used for detecting hybridization signal mistake or pollution；

So-called carrier material includes slide, sheet metal, silicon chip, nitrocellulose filter.

This detection type gene chip is characterised by that the clarithromycin drug resistance on design alternative helicobacter pylori 23s gene is dashed forward Conjugate the mediated quinolone resistance sudden change on wild type corresponding for point A2142G with A2143G and saltant type, helicobacter pylori gyrA gene Proton in wild type that site Asn87 (N87K) and Asp91 (D91G/Y/N) are corresponding and saltant type, human cytochrome enzyme CYP450 2C19*2 (G6981A) that pump inhibitor metabolism is relevant and the wild type of 2C19*3 (G636A) correspondence and saltant type totally 15 SNP Parting oligonucleotide probe.

Devising the many groups of amplimers to the high specific that above-mentioned target gene detects, its design principle is each Primer must be at the high degree of specificity position of Dui Ying target gene, and the amplification region of every pair of primer is the characteristic sequence of target gene； Primer include for chip detection needed for polymerase chain reaction (PCR), multiplex PCR, Standard PCR, asymmetric The specific primers such as PCR, every a pair specific primer can amplify corresponding target fragment.

The solid support material of these probes includes slide, sheet metal, silicon chip, nitrocellulose filter etc..

These surface of solid phase carriers with have double-active radical because of long chain organic compound modify, conventional having is double The long chain organic compound reagent of active group has: glutaraldehyde, trihydroxy methyl amino silane (APTES), N, N-diethoxy ammonia Base propyl-triethoxysilicane, poly-D-lysine, can use one therein or two kinds of combinations therein.

The PCR primer of amplification can be marked, also by mark fluorescent element Cy3, fluorescein Cy5, the primer of biotin DUTP or dCTP of the labellings such as fluorescein Cy3, fluorescein Cy5, biotin can be added in gene amplification process.

This detection type gene chip, can in the detection before helicobacter pylori infections " three furnace process " medicine for treatment, diagnose, monitor Middle application.Can be used for proton pump inhibitor metabolism, clarithromycin and the detection of mediated quinolone resistance situation.

We are consulting over the years on the basis of lot of documents, obtain people CYP2C19*2, CYP2C19*3 from GeneBank With the nucleotide sequence of helicobacter pylori, Clustalx1.8.msw Alignment software is used to carry out sequence analysis analysis, Determining relative conserved region, the analysis combining the dialect PCR sequencing primer designs such as Primer Premier5.0 and assessment is soft Part, the blast program reapplying NCBI screens, and requires and primer screening requirement in conjunction with amplification, has carried out PCR primer and set Meter.

In gene chip application probe be nucleic acid molecular probe, be the nucleic acid fragment of known particular sequence, can with mutually The nucleotide sequence generation molecule hybridization mended, therefore can utilize probe hybridization technique to detect the feature sheet of specific gene in sample Section and or the sequence of the gene that locates.For the characteristic fragments of target gene group, the sequence polymorphism of chromosomal DNA, gene The site of variation and feature etc., design and select suitable nucleic probe and make chip.By with sample in extract nucleic acid (DNA) hybridization, a step detection is obtained with the metabolic information of people CYP450 metabolism related gene 2C19*2,2C19*3 and imprisons The Helicobacter pylori drug resistance information to clarithromycin and quinolones.

Source according to nucleic acid molecular probe and character thereof can be divided into genome DNA probe, cDNA probe, rna probe, Peptide nucleic acid probe and oligonucleotide probe etc..The advantage of oligonucleotide probe be can as desired to indiscriminately ad. as one wishes adjust and Synthesize corresponding sequence, it is to avoid highly repetitive sequence adverse effect present in natural acid probe.Oligonucleoside Acid probe has the following characteristics that probe length is generally 10～50bp, and owing to the complexity of sequence is low, needed for hybridization time Between shorter.Oligonucleotide probe can be also used for detecting the variation of Individual base, analyzes for single nucleotide polymorphism (SNP).

We have employed the method for synthetic oligonucleotide probe.The correctness that probe selects, it will directly affect hybridization The analysis of result.What selection probe was most basic should have high degree of specificity in principle, considers Tm value, base number and making simultaneously The factor such as convenience.

The principle of probe design is as follows:

(1) specificity highly and sensitivity: the probe filtered out should be positioned at the height in primer amplified sequence Conserved region, to ensure specificity and the sensitivity of probe；Meanwhile, the probe of screening should be to other antibacterials and microorganism, relevant In the genome sequence of animal, environment, frequently seen plants gene order homology as far as possible is low, to avoid false positive results；Typing is visited Pin no cross reaction.Find and filter out the probe of high degree of specificity, this it is critical that.

(2) the Tm value of probe is the most consistent: because to carry out the hybridization of multiple probe on a chip simultaneously, At a certain temperature, the most each probe Tm value is most important, if Tm value is inconsistent, gap is excessive, and the signal after hybridization is strong Weak meeting is affected by suitable.General Tm value difference is less than three degree of ratios conveniently.

(3) secondary structure avoided as far as possible by the probe screened: probe one be preferably no hairpin structure, self dimer, Mispairing etc..Otherwise easily form the result of mistake.

(4) avoid the sequence choosing C, G rich region to make probe as far as possible.

(5) it is formed without dimer and mispairing etc. between the complementary series of each probe.

Recalling required gene order from Gene Bank, relatively same target-gene sequence is with or without the situation that there is variation.Really After target fragment to be expanded and primer, the principle designed according to above-mentioned probe, with biologies such as Primer Primer5.0 Learn software and search out all possible probe in PCR amplification region on the base alignment figure of required gene, in conjunction with The homology analysis softwares such as Clustalx1.8.mswAlignment are found out the height in particular target gene order amplification region and are protected Defending zone and respective specific sequence, the high conservative region in specific amplified fragments, and there is genus and species specificity structure Sequence is as typing probes.Log in U.S.'s Biotechnology Information center (National Center for Biotechnology Information, NCBI) homepage (http://www.ncbi.nlm.nih.gov) select nucleotide sequence BLAST, count According to the similar retrieval in storehouse, the probe of screening and other common bacteria known and microorganism, relevant animal in nucleic acid amplification region Genome sequence, frequently seen plants gene order homology as far as possible is low in environment, Tm value approximation on the average 60, G+C content is about 50%, according to Blast result, in conjunction with demand and experience, by hand the probe sifted out is finely adjusted and repairs.Actual below Chip hybridization detection in, need and testing result according to overall, have adjusted the probe that part uses.

The carrier selecting aldehyde group modified microscope slide to be chip, slide is domestic high-quality aldehyde radical sheet.

The probe of synthesis is dissolved in special sampling liquid, uses chip point sample instrument point sample.By right to 15 probes, 1 feminine gender According to probe and one sample application array of a positive quality control probe design forming, each slide has 10 each identical sample application arrays.Remove Outer every probe points 3 each point of positive quality control probe, in array 8 positive quality control probe points of parallel above point.Point sample is complete, chip Normal temperature drying saves backup.

For the oligonucleotide not being combined with aldehyde radical on eluting chip, by chip successively with eluent and ddH₂After O rinses, Can come into operation after natural drying.

We collect sample, and test and comparison also optimizes the processing method of various tested sample, establishes rapid extraction high The S.O.P. of quality target DNA fragment.Identical processing method taked by identical sample as far as possible.For required fragment base Cause, devises corresponding PCR amplification method: at 95 DEG C, degeneration 15 minutes；Then 95 DEG C, 30 seconds；65 DEG C, 1min；So run 44 circulations；Last at 72 DEG C, extend 5 minutes.Hybridization and washing methods: PCR primer is mixed with hybridization solution, is added to described Wash after hybridizing 1 hour in 45 DEG C of water-baths on chip；It is subsequently adding Streptavidin-Nanogold37 DEG C of reaction Wash after 30min, put room temperature and dry.Coloration method: silver staining reagent A liquid and B liquid equal-volume are mixed, is added on chip, treats core Stop colour developing after macroscopic Lycoperdon polymorphum Vitt or black round dot occur on sheet, clean with deionized water, dry.

Test result indicate that, the helicobacter pylori infections individualized treatment detection chip of preparation there are no nonspecific friendship Fork reaction.Multiple sample is detected, all shows preferable specificity and sensitivity.With sequencing technologies and fluorescent quantitation PCR method is compared, the method can once experiment in analyze simultaneously helicobacter pylori clarithromycin, mediated quinolone resistance site and The gene pleiomorphism of PPI metabolism relevant people CYP2C19, improves detection efficiency.Meanwhile, the accuracy of the method and sequencing technologies Identical, detection sensitivity is then significantly larger than gene sequencing technology, it is possible to the helicobacter pylori that patient's stomach is a small amount of detected, Initial infection finds antibacterial, in order to take corresponding remedy measures in early days.Compared with cultivating detection technique with traditional susceptibility, can Result accurately and reliably, the 6～7 of relative susceptibility cultivation can be provided in 6 hours without cultivation depending on changing gene chip detecting technique My god, the waiting time of staff and patient can be saved in a large number, strive for valuable time for treatment in time.

Advantages of the present invention

(1) high specific: the specificity of chip detection is through Double Selection, consistent with sequencing result specificity；

(2) high throughput testing and the Parallel testing of Multi-example to sample: can simultaneously provide people CYP450 metabolism related Metabolic information and the helicobacter pylori drug resistance information to clarithromycin and quinolones because of 2C19*2,2C19*3；

(3) high sensitivity and reliability: it is new that detection chip is molecular hybridization and biotin labeling technology combines Type molecular diagnosis method, and when template and probe hybridize, reaction condition and reaction volume are completely the same, eliminate and test Artificial and other error in journey；

(4) the detection time is short, and consumption sample amount is few, and reaction volume is little, is suitable for the needs of Clinical detection；

(5) favorable reproducibility.

Accompanying drawing explanation

The probe array figure that Fig. 1: helicobacter pylori infections individualized treatment detection gene chip is final.A round dot in figure Represent a point sample of probe, 3 times that 3 round dots are probe repetition point samples of vertical direction.Round dot in dotted line frame is 15 Species specific probes, the last item is blank.Round dot outside dotted line frame is identity column.

Fig. 2: helicobacter pylori infections individualized treatment detection gene chip Evaluation on specificity result figure.In figure, 1 is carat Mycin 2142W-2143W, quinolinones 87-WC, quinolinones 91-MG；2 is clamycin 2 142W-2143M, quinolinones 87-MG, quinoline Promise ketone 91-W；3 clamycin 2 142M-2143W, quinolinones 87-MA, quinolinones 91-W；4 is clamycin 2 142W-2143M, Quinolinones 87-WT, quinolinones 91-MA；5 is CYP450-2C19*2W, CYP450-2C19*3W；6 is CYP450-2C19*2M, CYP450-2C19*3M。

Fig. 3: helicobacter pylori infections individualized treatment detection gene chip sensitivity technique analysis result figure.With pylorus spiral shell Bacillus drug resistant gene detection site clamycin 2 142W-2143M, quinolinones 87-WT, quinolinones 91-MA and human genome As a example by CYP4502C19*2-W, CYP4502C19*3-W, wherein in figure, A-I represents its sensitivity evaluation result.A-I represents successively Helicobacter pylori 102CFU/ml, 103CFU/ml, 104CFU/ml, 105CFU/ml, resistive comparison, human genome 10ng/ μ l, 5ng/ μ l, 2ng/ μ l, negative control.

Detailed description of the invention

A kind of helicobacter pylori infections individualized treatment detection gene chip, includes process and the amplification of template；Few core Thuja acid probe and solidification thereof；Detection and analysis etc..

Embodiment one: the preparation of a kind of helicobacter pylori infections individualized treatment detection gene chip

1. prepare special primer

Select helicobacter pylori 23s gene clarithromycin resistant mutational site A2142G and A2143G；Helicobacter pylori Mediated quinolone resistance catastrophe site Ash87, Asp91 on gyrA gene；Select proton pump suppression in human cytochrome enzyme CYP450 2C19*2 (G6981A) and 2C19*3 (G636A) conduct that agent metabolism is relevant detect target

Mark carries out specific primer design (being shown in Table 1).All downstream primers 5 ' end labelling biotin.

The amplification target gene species of table 1 specific primer and correspondence thereof

2. design and screen people's proton pump inhibitor metabolism related locus and Hp Drug Resistance site probe

Wild type and saltant type SNP is separately designed for A2142G, A2143G, N87K, D91G/Y/N, G681A, G636A Typing probes (as shown in table 2).Design chip base Quality Control probe, negative control and positive control probe simultaneously.All oligonucleotide Probe 3 ' section carries out amido modified.

Table 2 specific probe sequence and the target gene of correspondence

3. prepare oligonucleotide chip

Dissolve the oligonucleotide probe lyophilized powder of synthesis adds suitable sterile water for injection, use deionized water conduct Blank, UV surveys concentration, after reading OD260 numerical value, calculates concentration and probe concentration by 1OD=33 μ g/ml, by each probe sterilizing Water for injection is diluted to 100 μMs, stand-by.During point sample, oligonucleotide probe 2 × sampling liquid (6 × SSC, 0.1%SDS) dilutes To final concentration 50 μMs, take 20 μ l and be transferred to 384 orifice plates.With gene chip sample applying instrument, probe points is modified glass to blank aldehyde radicalization On sheet, keeping temperature in point sample instrument is 23 DEG C, and relative humidity is more than 85%.After oligonucleotide chip preparation, before using extremely Few placement in room temperature is dried 18 hours, stand-by.Specific Oligonucleotide Probes for Microarray Detection array includes in human cytochrome enzyme P450 respectively 2C19*2 (G6981A) that proton pump inhibitor metabolism is relevant and 2C19*3 (G636A) and Hp Drug Resistance detect probe, Its probe array of arranging on chip is as shown in table 3.

Table 3 oligonucleotide probe array

Chip base Quality Control

Chip base Quality Control

Chip base Quality Control

Chip base Quality Control

Chip base Quality Control

Chip base Quality Control

Chip base Quality Control

Chip base Quality Control

2C192W

2C192M

2C193W

2C193M

42W43W

42M43W

42W43M

42M43M

2C192W

2C192M

2C193W

2C193M

42W43W

42M43W

42W43M

42M43M

2C192W

2C192M

2C193W

2C193M

42W43W

42M43W

42W43M

42M43M

87WT

87WC

87MA

87MG

91W

91MG

91MA

H2O

87WT

87WC

87MA

87MG

91W

91MG

91MA

H2O

87WT

87WC

87MA

87MG

91W

91MG

91MA

H2O

4. extract helicobacter pylori and human gene group DNA

Being sucked by gastric mucosa in clean 0.5ul EP pipe, (article No.: S070001, Shenzhen is auspicious to add 50ul DNA extraction liquid Health Bioisystech Co., Ltd), 100 DEG C are boiled 10min, place 30min for 4 DEG C；Then room temperature 13000rpm is centrifuged 2min, takes Putting into clearly in new EP pipe ,-20 DEG C frozen standby.

5. preparation detection reference material

In order to screen proton pump inhibitor metabolism related locus and Hp Drug Resistance abrupt climatic change probe, by people Check order after genomic DNA and Helicobacter pylori Strains DNA cloning, be prepared for human gene group DNA's reference material of 4 types and 4 kinds Type helicobacter pylori DNA reference material.Every kind of reference material gradient dilution prepares sensitivity reference material.

6. set up and optimize PCR system

PCR system is a pipe quadruple asymmetric PCR system.Suitably PCR system can improve chip detection further Sensitivity.The factor such as consumption of labeled primer and the absolute concentration of non-marked primer and relative scale, Taq enzyme has been carried out excellent Change.Final concentration of 0.12 μM: 1.2 μMs of 2C19*2 upstream and downstream primer；Final concentration of 0.12 μM: 1.2 μMs of 2C19*3 upstream and downstream primer, 23s upstream and downstream primer final concentration of 0.1 μM: 0.1Mm；Final concentration of 0.2 μM: 0.2 μ of 23s, gyrA upstream and downstream primer When MHotMasterTaq DNA Polymcrase enzyme dosage is 2.5U/ system, the probe gray value of reference material is relatively strong, and pylorus Still can detect when Helicobacter pylori low copy template 103CFU/ml, human genome low copy template 2ng/ μ l.Finally determine PCR system is as shown in table 4.Preferably PCR amplification condition is: 95 DEG C, degeneration 15 minutes；Then 95 DEG C, 30 seconds；65 DEG C, 1min； Run 44 circulations；Last at 72 DEG C, extend 5 minutes.

Table 4PCR system formulation

7. set up and optimize hybridization system

Suitably hybridization system also has great role to specificity and the sensitivity improving of chip.Obtained same by optimization Time can ensure that hybridization solution composition, hybridization conditions and the post-hybridization wash conditions of specificity and sensitivity.PCR in hybridization system Product mixes with hybridization solution equal-volume, each composition of hybridization solution final concentration of 4 × SSC, 0.3%SDS, 5% Methanamide, 16 μMs of 20T- NH2.Hybridization conditions is that 45 DEG C of water-baths hybridize 1 hour.Wash conditions is washing liquid A (1 × SSC, 0.2%SDS), washing liquid B under room temperature (0.2 × SSC) and washing liquid C (0.1 × SSC) respectively wash 20s.

8. prepare Visual retrieval reagent and set up coloration method

Add by the Streptavidin-Nanogold10 μ l of 1: 40 dilution proportion, diluent in each hybridization region of chip Composition: 1 × PBS+0.1%BSA, places 30min for 37 DEG C；Clean 20s by PBST washing liquid after taking-up, in triplicate, use deionization Water rinses, and puts room temperature and dries.After by silver staining reagent A liquid (aqueous solution of silver acetate, concentration 4mg/mL) and the B liquid (Fructus Citri Limoniae of hydroquinone Acid buffer solution, concentration 10mg/mL) equal-volume mixing, each hybridization region lucifuge immediately adds A, B mixed liquor of 30ul, treats Stop colour developing after macroscopic Lycoperdon polymorphum Vitt or black round dot occur on chip, clean with deionized water, dry.

Embodiment 2: the determination of gene chip Positive judgement standards

All results judge to be as the criterion with the signal value of computed in software, and what signal value reflected is the gray value of probe.In chip Chip base Quality Control point, negative Quality Control point and human genome and the positive value equal root of interpretation standard of helicobacter pylori each drug resistance site probe Method of learning according to statistics is calculated.Two probes (2C19*2-W and 2C19*2M) for CYP450-2C19*2 are visited when a certain bar The average gray value of pin be another twice and above time, then judge that this probe is as positive；When both average gray value ratios When value is less than 2, then judge that this site is heterozygosis.Two probe (2C19*3-W and 2C19*3M) interpretation marks of CYP450-2C19*3 Standard is same as CYP450-2C192.For clarithromycin drug resistance site 4 probes (42W-43W, 42M-43W, 42W-43M and 42M-43M) when a certain bar probe the twice that average gray value is another three and above time, then judge that this probe is as positive. Four probe (87WT, 87WC, 87MA and 87MG) interpretation standard for mediated quinolone resistance site 87 are same as clarithromycin drug resistance The probe in site.Carat is also same as three probe (91W, 91MG and 91MA) interpretation standard in mediated quinolone resistance site 91 mould The probe in element drug resistance site.

Embodiment 3: helicobacter pylori infections individualized treatment detection gene chip Evaluation on specificity

Specificity is the most important performance assessment criteria of diagnostic method, and the present invention uses the system and condition optimized, have detected The bacterial strain of various common variation situations and human genome, chip detection result is shown in accompanying drawing 2.As seen from the figure, the present invention is utilized Can the bacterial strain of common variation situation and human genome correctly be distinguished, specificity is good.

Embodiment 4: helicobacter pylori infections individualized treatment detection gene chip sensitivity evaluation

Respectively detect the sensitivity of probe to evaluate chip, we are by 4 strain helicobacter pylorus of the different genotype through cultivating The human genome target gene plasmid of bacteria strain and structure is as reference material.With human gene group DNA as template, use the primer of correspondence (downstream primer is labelling) carries out RT-PCR amplification, and product is connected to PGM-Tvector through using T4DNA ligase after purification (TIANGEN), transformed competence colibacillus bacillus coli DH 5 alpha (TIANGEN), extract plasmid with plasmid extraction kit (TIANGEN), Correct through sequence verification recombiant plasmid sequence.It is prepared for 4 kinds of human genome plasmid DNA reference materials containing target gene altogether.By nothing The water of RNase enzyme is the most stand-by after dissolving corresponding Helicobacter pylori Strains and human genome plasmid respectively, subpackage in-70 DEG C Preserve, as DNA reference material.Every kind of DNA reference material gradient dilution prepares sensitivity reference material.

Use Helicobacter pylori Strains and human genome plasmid sensitivity reference material that the detection sensitivity of chip is carried out Evaluate, found that chip is all able to detect that the plasmid DNA of 103CFU/ml system to every kind of bacterial strain, to human gene group DNA's energy The plasmid DNA of 2ng/ μ l system enough detected.With clamycin 2 142W-2143M, quinolinones 87-WT, quinolinones 91-MA and people Genome C YP4502C19*2-W, as a example by CYP4502C19*3-W, select 102CFU/ml, 103CFU/ml, 104CFU/ml, The helicobacter pylori of 105CFU/ml and 10ng/ μ l, 5ng/ μ l, 2ng/ μ l human gene group DNA and negative control carry out chip inspection Surveying, result is shown in accompanying drawing 3, and as seen from the figure, chip is able to detect that helicobacter pylori and the 2ng/ μ l plasmid of 103CFU/ml DNA。

In addition to the implementation, the present invention also has other embodiments.Every employing equivalent or equivalent transformation are formed Technical scheme, all in the protection domain of application claims.

Claims (2)

1. a helicobacter pylori infections individualized treatment detection gene chip, it is characterised in that chip includes (1) helicobacter pylorus Wild type corresponding for clarithromycin resistant mutational site A2142G with A2143G and saltant type SNP typing on bacterium 23s gene are few Nucleotide probe；(2) open country corresponding for mediated quinolone resistance mutational site N87K with D91G/Y/N on helicobacter pylori gyrA gene Raw type and saltant type SNP parting oligonucleotide probe；(3) in human cytochrome enzyme CYP450, proton pump inhibitor metabolism is relevant The wild type corresponding for G636A of G6981A with 2C19*3 of 2C19*2 and saltant type SNP parting oligonucleotide probe；(4) chip Quality control oligonucleotide probe；(5) oligonucleotide probe solidifies the probe array formed on a support material；

It is metabolism related that so-called helicobacter pylori infections individualized treatment detection gene chip refers to detect people CYP450 simultaneously Because the gene type of 2C19*2,2C19*3 and helicobacter pylori are to the Resistant genetype of clarithromycin and quinolones Oligonucleotide arrays；

So-called oligonucleotide probe refers to and the poly oligonucleotide of genes of interest hybridization, ten to five ten bases of length；

So-called quality control oligonucleotide probe at least includes two kinds of probes, i.e. positive controls, negative control: positive control is used In detecting whether normal hybridisation and being accurately positioned, negative control is used for detecting hybridization signal mistake or pollution；

So-called carrier material is selected from slide, sheet metal, silicon chip, nitrocellulose filter；

15 SNP parting oligonucleotide probes that SNP typing is corresponding are as shown in the table:

Table 1. specific probe sequence

2. the preparation method of helicobacter pylori infections individualized treatment detection gene chip as claimed in claim 1, its feature It is to comprise the following steps:

1) design SNP typing probes: with helicobacter pylori 23s gene and gyrA gene and human cytochrome enzyme CYP450 gene For target, therefrom choose the probe sequence described in claim 1；

2) synthesising probing needle: use DNA synthesizer synthesis；

3) probe processes: at 3 ' or 5 ' ends plus polyT tail, and carries out amido modified, enables it to be solidificated in substrate；

4) preparation of gene chip: use point sample instrument to carry out point sample by pre-set order in substrate；

5) chip put is placed dry more than 18 hours in room temperature, make probe fixedly secure in substrate.